Cloning And Sequence Analysis Of Equine Infectious Anemia Virus WH17 Strain And Study On Promoter Activity Of Long Terminal Repeat

Equine infectious anemia virus (EIAV), a member of lentivirus family which related with human immunodeficiency virus, causes a life-long infection and chronic disease in horses characterized by periodic episodes of fever, plasma viremia, anemia, and thrombocytopenia. The EIAV proviral genomic long-terminal repeat (LTR) is a eukaryotic promotor, which plays an important regulatory role on transcription and replication of the virus.The EIAV provirus DNA of the strain isolated from a naturally infected horse was amplified by PCR in four fragments, the PCR product of each fragment was cloned into pMD18-T vector, the recombinants identified by PCR and enzyme digestion were sequenced, and the positive clones of the four fragments were named as p2.8, p2.4, p2.6 and p1.2, respectively, then the complete nucleotide sequence of the EIAV provirus DNA was obtained by splicing the four fragments. The genome of the isolated strain was 8268bp in length and the sequence of which was 86.0%,85.8%and 85.2% in homology with those of L-EIAV,DA-EIAV and DLA-EIAV respectively by using DNAStar.By comparing the sequence of LTR between WH17 strain and L,DA,DLA strain, we found that, there is one E-box motif in U3 and R conjunction region. So it is assumed that the E-box might be associated with the enhancement of transcription activity.Here, the LTRs of WH17 ,DLA-25,DLA-118 and LTR with single mutation in the E-box motif were separately cloned into pCAT-Basic vector resulting in a series of recombinant plasmids containing CAT reporter under the control of different versions of LTRs. Standard transactivition assays utilizing the EIAV LTR- driven CAT gene were performed in fetal donkey dermal (FDD) cell,donkey leucocytes(DL), EIAV-infected FDD cell and EIAV-infected DL ,respectively. The results indicated that Tat can specifically increase the promoter activity of LTR from EIAV-DLA, cannot increase that of LTR with single mutation in the E-box motif in U3 and R conjunction region;we didn't find promoter activity of LTR in fetal donkey dermal (FDD) cell, suggesting that the E-box motif in U3 and R conjunction region cannot promote transcription activity of LTR from WH17 isolate strain, cellular origin of LTR is related to Tat trans-activation, long terminal repeat is associated with cell tropism.